Reaction of thymidine and ascorbic acid induced by UV in the presence of salicylic acid.

When a neutral solution of thymidine and ascorbic acid was irradiated with UV light of wavelength longer than 300 nm in the presence of salicylic acid as a photosensitizer, six product peaks appeared in an HPLC chromatogram in addition to small amounts of thymidine dimers. The six products were identified as three pairs of diastereomers of 5-(2-deoxy-2-l-ascorbyl)-5,6-dihydrothymidine, 5-(2-l-ascorbyl)-5,6-dihydrothymidine, and 5,6-dihydrothymidine. These results suggest that novel DNA damage may be generated by ascorbic acid with salicylic acid induced by sunlight.

El extracto acuoso de Cymbopogon citratus protege al ADN plasmídico del daño inducido por radiación UVC / Cymbopogon citratus aqueous extract protects plasmid DNA from UVC-induced damage

Objetivo: Evaluar el efecto protector del extracto acuoso de Cymbopogon citratus (DC) Stapf, ante el daño inducido por las radiaciones UVC. Material y Métodos: Para evaluar si el extracto acuoso de C. citratus era capaz de inducir roturas de cadenas en el ADN, moléculas de plásmido pBluescript SK II fueron tratadas con diferentes concentraciones del extracto (0,01 - 4,0 mg/mL), en los tiempos de exposición: 30, 60 y 90 min. El efecto fotoprotector fue evaluado aplicando el extracto vegetal antes, durante, y después de la irradiación del ADN plasmídico con 200 J/m2 de UVC. La actividad enzimática de T4 endonucleasa V fue empleada para detectar formación de CPDs. Las formas superenrollada y relajada de las moléculas de plásmido fueron separadas electroforéticamente en gel de agarosa. Adicionalmente, se midió la transmitancia del extracto acuoso a la DO de 254 nm. Resultados: Ninguna de las concentraciones evaluadas resultó genotóxica con 30 min de tratamiento. Las concentraciones ≥ 2 mg/mL indujeron roturas de cadenas a los 90 min de incubación. El extracto de C. citratus a concentraciones ≥ 0,5 mg/mL protegió al ADN frente a las radiaciones UVC. Conclusiones: En nuestras condiciones experimentales, el extracto acuoso de C. citratus protege al ADN frente a la genotoxicidad inducida por la luz UVC, previniendo la generación de CPDs, pero no es capaz de eliminarlas una vez formadas

Aim: to evaluate the photoprotective effect of aqueous extract of Cymbopogon citratus (DC) Stapf against UVC-induced damage to ADN. Material and methods: In the experimental procedure, samples of plasmid pBluescript SK II solutions were exposed to C. citratus aqueous extract in 0.01-4.0 mg/mL concentrations during 30, 60 and 90 min. In order to evaluate the photoprotective effect, the vegetal extract was applied before, during and after UVC radiation at 200 J/m2 doses. DNA repair enzymes T4 endonuclease V was employed in order to discriminate CPDs damage. Then, supercoiled and relaxed forms of DNA were separated after electrophoretic migration in agarose gels. Also aqueous extract transmittance was measure at 254 nm OD. Results: None of the concentrations tested were genotoxic in 30 min of exposition. Concentrations ≥ 2 mg/mL induced strand breaks at 90 min of incubation. The C. citratus extract at concentrations ≥ 0.5 mg/ mL protect DNA in front of UVC radiation. Conclusions: In our experimental conditions, C. citratus extract protects DNA from the genotoxicity induced by light UVC, preventing the CPDs generation, but is not able to eliminate DNA damage once formed

Investigation of the mechanisms of photo-induced formation of cyclobutane dimers of cytosine and 2,4-diaminopyrimidine.

The mechanisms of the formation of cyclobutane dimers (CBD) of cytosine and 2,4-diaminopyrimidine were studied at the CC2 theoretical level and cc-pVDZ basis functions. Four orientations of the two monomers are explored: cys-syn, cis-anti, trans-syn, and trans-anti. The research revealed that in all cases the cyclobutane structures are formed along the (1)ππ* excited-state reaction paths of the stacked aggregates. We localized the S1/S0 conical intersections mediating those transformations. The results obtained agree well with the previously reported investigations on the cis-syn cyclodimer formations of other pyrimidines.

Blocking cyclobutane pyrimidine dimer formation by steric hindrance.

The efficiency of thymine (Thy) and uracil (Ura) to form cyclobutane pyrimidine dimers (CPDs) in solution, upon UV irradiation differs by one order of magnitude. This could to be partially related to the steric hindrance induced by the methyl at C5 in thymine. The aim of the present work is to establish the influence of a bulky moiety at this position on the photoreactivity of pyrimidines. With this purpose, photosensitization with benzophenone and acetone of a 5-tert-butyl uracil derivative () and the equivalent Thy () has been compared. Introduction of the tert-butyl group completely blocks CPD formation. Moreover, the mechanistic insight obtained by laser flash photolysis is in accordance with the observed photoreactivity.

Preparation of oligodeoxyribonucleotides containing the pyrimidine(6-4)pyrimidone photoproduct by using a dinucleotide building block.

This unit describes procedures for the synthesis of a dinucleotide-type building block of the pyrimidine(6-4)pyrimidone photoproduct [(6-4) photoproduct], which is one of the major DNA lesions induced by ultraviolet (UV) light, and its incorporation into oligodeoxyribonucleotides. Although this type of lesion is frequently found at thymine-cytosine sites, the building block of the (6-4) photoproduct formed at thymine-thymine sites can be synthesized much more easily. The problem in the oligonucleotide synthesis is that the (6-4) photoproduct is labile under alkaline conditions. Therefore, building blocks with an amino-protecting group that can be removed by a brief treatment with ammonia water at room temperature must be used for the incorporation of the normal bases. Byproduct formation by the coupling of phosphoramidites with the N3 of the 5' component should also be considered. This side reaction can be avoided by using benzimidazolium triflate as an activator.

Expanding the horizon of the thymine isostere biochemistry: unique cyclobutane dimers formed by photoreaction between a thymine and a toluene residue in the dinucleotide framework.

Substituted toluenyl groups are considered as close isosteres of the thymine residue. They can be recognized by DNA polymerases as if they were thymine. These toluene derivatives are generally inert toward radical additions, including the [2+2] photo-cycloadditions, due to the stable structure of the aromatic ring and are usually used as solvents for radical reactions. Surprisingly, after incorporating toluene into the dinucleotide framework, we found that the UV excited thymine residue readily dimerizes with the toluenyl moiety through a [2+2] photo-addition reaction. Furthermore, the reaction site on the toluenyl moiety is not the C5=C6 bond, as commonly observed in cyclobutane pyrimidine dimers, but the C4=C5 or C3=C4 instead. Such a reaction pattern suggests that in the stacked structure, it is one of these bonds, not the C5=C6, that is close to the thymine C5=C6 bond. A similar structural feature is found in DNA duplex with a thymine replaced by a 2,4-difluorotoluene. Our results argue that although the substituted toluenyl moieties closely mimic the size and shape of the thymine residue, their more hydrophobic nature determines that they stack on DNA bases differently from the natural thymine residue and likely cause local conformational changes in duplex DNA.

Photosensitized [2 + 2] cycloaddition of N-acetylated cytosine affords stereoselective formation of cyclobutane pyrimidine dimer.

Photocycloaddition between two adjacent bases in DNA produces a cyclobutane pyrimidine dimer (CPD), which is one of the major UV-induced DNA lesions, with either the cis-syn or trans-syn structure. In this study, we investigated the photosensitized intramolecular cycloaddition of partially-protected thymidylyl-(3'→5')-N(4)-acetyl-2'-deoxy-5-methylcytidine, to clarify the effect of the base modification on the cycloaddition reaction. The reaction resulted in the stereoselective formation of the trans-syn CPD, followed by hydrolysis of the acetylamino group. The same result was obtained for the photocycloaddition of thymidylyl-(3'→5')-N(4)-acetyl-2'-deoxycytidine, whereas both the cis-syn and trans-syn CPDs were formed from thymidylyl-(3'→5')-thymidine. Kinetic analyses revealed that the activation energy of the acid-catalyzed hydrolysis is comparable to that reported for the thymine-cytosine CPD. These findings provided a new strategy for the synthesis of oligonucleotides containing the trans-syn CPD. Using the synthesized oligonucleotide, translesion synthesis by human DNA polymerase η was analyzed.

Excited-state energies and electronic couplings of DNA base dimers.

The singlet excited electronic states of two pi-stacked thymine molecules and their splittings due to electronic coupling have been investigated with a variety of computational methods. Focus has been given on the effect of intermolecular distance on these energies and couplings. Single-reference methods, CIS, CIS(2), EOM-CCSD, TDDFT, and the multireference method CASSCF, have been used, and their performance has been compared. It is found that the excited-state energies are very sensitive to the applied method but the couplings are not as sensitive. Inclusion of diffuse functions in the basis set also affects the excitation energies significantly but not the couplings. TDDFT is inadequate in describing the states and their coupling, while CIS(2) gives results very similar to EOM-CCSD. Excited states of cytosine and adenine pi-stacked dimers were also obtained and compared with those of thymine dimers to gain a more general picture of excited states in pi-stacked DNA base dimers. The coupling is very sensitive to the relative position and orientation of the bases, indicating great variation in the degree of delocalization of the excited states between stacked bases in natural DNA as it fluctuates.

Role of adenine in thymine-dimer repair by reduced flavin-adenine dinucleotide.

We present a study of excited-state behavior of reduced flavin cofactors using femtosecond optical transient absorption spectroscopy. The reduced flavin cofactors studied were in two protonation states: flavin-adenine dinucleotide (FADH2 and FADH-) and flavin-mononucleotide (FMNH2 and FMNH-). We find that FMNH- exhibits multiexponential decay dynamics due to the presence of two bent conformers of the isoalloxazine ring. FMNH2 exhibits an additional fast deactivation component that is assigned to an iminol tautomer. Reduced flavin cofactors also exhibit a long-lived component that is attributed to the semiquinone and the hydrated electron that are produced in photoinduced electron transfer to the solvent. The presence of adenine in FADH2 and FADH- further changes the excited-state dynamics due to intramolecular electron transfer from the isoalloxazine to the adenine moiety of cofactors. This electron transfer is more pronounced in FADH2 due to pi-stacking interactions between two moieties. We further studied cyclobutane thymine dimer (TT-dimer) repair via FADH- and FMNH- and found that the repair is much more efficient in the case of FADH-. These results suggest that the adenine moiety plays a significant role in the TT-dimer repair dynamics. Two possible explanations for the adenine mediation are presented: (i) a two-step electron transfer process, with the initial electron transfer occurring from flavin to adenine moiety of FADH-, followed by a second electron transfer from adenine to TT-dimer; (ii) the preconcentration of TT-dimer molecules around the flavin cofactor due to the hydrophobic nature of the adenine moiety.

Structure determination of an interstrand-type cis-anti cyclobutane thymine dimer produced in high yield by UVB light in an oligodeoxynucleotide at acidic pH.

UVB irradiation of DNA produces photodimers in adjacent DNA bases and on rare occasions in nonadjacent bases. UVB irradiation (312 nm) of d(GTATCATGAGGTGC) gave rise to an unknown DNA photoproduct in approximately 40% yield at acidic pH of about 5. This product has a much shorter retention time in reverse phase HPLC compared to known dipyrimidine photoproducts of this sequence. A large upfield shift of two thymine H6 NMR signals and photoreversion to the parent ODN upon irradiation with 254 nm light indicates that the photoproduct is a cyclobutane thymine dimer. Exonuclease-coupled MS assay establishes that the photodimer forms between T2 and T7, which was confirmed by tandem mass spectrometric MS/MS identification of the endonuclease P1 digestion product pd(T2[A3])=pd(T7[G8]). Acidic hydrolysis of the photoproduct gave a product with the same retention time on reverse phase HPLC and the same MS/MS fragmentation pattern as authentic Thy[ c,a]Thy. 2D NOE NMR data are consistent with a cis-anti cyclobutane dimer between the 3'-sides of T2 and T7 in anti glycosyl conformations that had to have arisen from an interstand type reaction. In addition to pH dependency, the photoproduct yield is highly sequence specific and concentration dependent, indicating that it results from a higher order folded structure. The efficient formation of this interstrand-type photoproduct suggests the existence of a new type of folding motif and the possibility that this type of photoproduct might also form in other folded structures, such as G-quadruplexes and i-motif structures which can be now studied by the methods described.

Computational study of thymine dimer radical anion splitting in the self-repair process of duplex DNA.

Formation of the thymine dimer is one of the most important types of photochemical damage in DNA, responsible for several biological pathologies. Though specifically designed proteins (photolyases) can efficiently repair this type of damage in living cells, an autocatalytic activity of the DNA itself was recently discovered, allowing for a self-repair mechanism. In this paper, we provide the first molecular dynamics study of the splitting of thymine dimer radical anions, using a quantum mechanical/molecular mechanics (QM/MM) approach based on density functional theory (DFT) to describe the quantum region. A set of seven statistically representative molecular dynamics trajectories is analyzed. Our calculations predict an asynchronously concerted process in which C5-C5' bond breaking is barrierless while C6-C6' bond breaking is characterized by a small free energy barrier. An upper bound of 2.5 kcal/mol for this barrier is estimated. Moreover, the molecular dynamics study and the low free energy barrier involved in C6-C6' bond breaking characterize the full process as being an ultrafast reaction.

Electronic coupling in the excited electronic state of stacked DNA base homodimers.

The nature of the electronic coupling of stacked nucleic acid bases adenine (A), thymine (T), and cytosine (C), in A-A, T-T, and C-C complexes in their excited states was investigated; a different character of the electronic coupling for the T-T complex was shown.

Production of cis-syn thymine-thymine cyclobutane dimer oligonucleotide in the presence of acetone photosensitizer.

cis-syn Cyclobutane pyrimidine dimer (CPD) oligonucleotide was produced by UV irradiation in the presence of acetone photosensitizer. Acetone could enhance the productivity but evidently induced the photocleavage of oligonucleotide under a long time irradiation. A statistical approach of orthogonal design was applied to optimize the preparation condition for the production of the modified oligonucleotide. Optimal conditions for maximal cis-syn CPD oligonucleotide productivity were determined based on three factors: acetone concentration, initial oligonucleotide concentration, and irradiation time at several different levels. The optimal modified oligonucleotide that this optimization could produce was 32.7%. Through analysis of 20% polyacrylamide gel electrophoresis, it was found that modified oligonucleotide migrated slightly more slowly than the parent oligonucleotide. The photoreactivation of cis-syn thymine-thymine dimer oligonucleotide displayed the selectivity of the substrate specificity of DNA photolyase with high-performance liquid chromatography (HPLC) analysis.

PNA-based reagents for the direct and site-specific synthesis of thymine dimer lesions in genomic DNA.

An acetophenone containing PNA-based reagent was designed for the direct and site-specific synthesis of a cis-syn thymidine dimer lesion in genomic DNA.

Optical detection of thymine dinucleoside monophosphate and its cis-syn photodimer by inorganic nanoparticles.

The Watson-Crick DNA double helix is an averaged ideal of multitudinous natural sequence-directed local structural deviations. By effectively derailing normal cellular physiological processes, damaged bases can induce noncanonical irregularities in the local structure of DNA if not efficiently repaired. Pyrimidine bases, especially thymine, are prone to dimerization when exposed to ultraviolet light. A [2 + 2] photocyclo-addition between adjacent thymine bases predominantly produces the cis-syn photodimer. These lesions, implicated in skin cancer, bend DNA by approximately 30 degrees due to their structural and conformational changes. Such changes in molecular properties can be detected by differential quenching of CdS nanoparticle luminescence and by surface-enhanced Raman scattering spectroscopy on metal nanoparticle substrates.

Assay method for Escherichia coli photolyase activity using single-strand cis-syn cyclobutane pyrimidine dimer DNA as substrate.

A high-performance liquid chromatography method for the assay of Escherichia coli photolyase activity was developed. When cis-syn cyclobutane pyrimidine dimer was used as substrate, the Michaelis constant (K(m)) value for the photolyase activity was 100 nM. The linear range of the calibration curve of the photolyase activity was 0.026-6.64 microU/assay tube. The correlation coefficient for this linearity was 0.998. The limit of detection (S/N = 3) was 26 nU/assay tube. The photolyase activity was increased 1.6-fold in the presence of 5,10-methenyltetrahydrofolic acid in the enzyme reaction mixture.

Chemical synthesis and translesion replication of a cis-syn cyclobutane thymine-uracil dimer.

The cytosine base in DNA undergoes hydrolytic deamination at a considerable rate when UV radiation induces formation of a cyclobutane pyrimidine dimer (CPD) with an adjacent pyrimidine base. We have synthesized a phosphoramidite building block of a cis-syn cyclobutane thymine-uracil dimer (T[]U), which is the deaminated form of the CPD at a TC site, and incorporated it into oligodeoxyribonucleotides. The previously reported method for synthesis of the thymine dimer (T[]T) was applied, using partially protected thymidylyl-(3'-5')-2'-deoxyuridine as the starting material, and after triplet- sensitized irradiation, the configuration of the base moiety in the major product was determined by NMR spectroscopy. Presence of the cis-syn cyclobutane dimer in the obtained oligonucleotides was confirmed by UV photoreversal and reaction with T4 endonuclease V. Using a 30mer containing T[]U, translesion synthesis by human DNA polymerase eta was analyzed. There was no difference in the results between the templates containing T[]T and T[]U and pol eta bypassed both lesions with the same efficiency, incorporating two adenylates. This enzyme showed fidelity to base pair formation, but this replication causes a C-->T transition because the original sequence is TC.

Sequence- and regioselectivity in the montmorillonite-catalyzed synthesis of RNA.

The possible role of catalysis in forming a limited number of RNAs from activated monomers is investigated by examining the sequence- and regioselectivity in the montmorillonite-catalyzed formation of RNA dimers and trimers. The reactivity of A was similar to that of G, and C was comparable in reactivity to U. Yet the reactivity of the purine nucleotides differed from that of the pyrimidines. In the reaction of nucleotides (pN) with activated monomers (ImpN), the sequence- and regioselectivity was Pu(3')Py > Pu(3')Pu = Pu(2')Py > Pu(2')Pu. The 5'-pyrimidine initiated dimers formed less efficiently than the 5'-purine initiated dimers. Trimer formation was investigated by the synthesis of 8 dimers (pNpN) and measuring the yields of trimers formed in the reaction of each dimer with a mixture of equal molar amounts of four activated monomers. The reactivity of the dimers depended on the nucleotide attached to the 3'-end of the RNA and the regiochemistry of the phosphodiester bond. Rules based on these studies are proposed to predict the sequence- and regioselectivity of the RNAs formed in montmorillonite-catalyzed reactions. These rules are consistent with the structures of the 2-5-mers formed in the reaction of equimolar amounts of ImpA and ImpC. This research establishes that the montmorillonite catalyst limits the number of RNA oligomer isomers formed. The potential significance of these findings to the origins of life is discussed.

Study on reactivity and protection of the alpha-hydroxyphosphonate moiety in 5'-nucleotide analogues: formation of the 3'-O-P-C(OH)-C4' internucleotide linkage.

The recently described epimeric nucleosidyl-5'-C-phosphonates (alpha-hydroxyphosphonates) represent novel nucleotide analogues that can be incorporated into chimeric oligonucleotides by the phosphotriester condensation method. In order to prepare suitable protected monomer(s) we have studied condensation reaction between protected 2'-deoxythymidine and 2'-deoxythymidinyl-5'-C-phosphonate, both as model compounds, in dependence on the nature of the 5'-hydroxyl protecting group. We have found that the O-acetyl group is unstable in the presence of TPSCl or MSNT used as condensing agents for activation of the phosphorus moiety. This instability negatively influences the scope of the condensation process. On the other hand, introduction of the O-methoxycarbonyl group gave excellent results. The O-methoxycarbonyl group does not participate in the condensation process, and its quantitative introduction into the nucleotide analo gues is accomplished using a novel acylating agent, methoxycarbonyl tetrazole.

Oligodeoxynucleotides containing amide-linked LNA-type dinucleotides: synthesis and high-affinity nucleic acid hybridization.

Synthesis of three different amide-linked LNA-type dinucleotides and their incorporation into 9-mer and 17-mer oligodeoxynucleotides is described; compared to the reference DNA-RNA duplex, incorporation of one of the three dimers (5'-DNA*LNA dimer) induced significantly increased duplex thermostabilities.